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PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
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| Zeitschriftentitel: | Scientific Reports |
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| Personen und Körperschaften: | , , , , , , , , |
| In: | Scientific Reports, 7, 2017, 1 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
Springer Science and Business Media LLC
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| Schlagwörter: |
| Zusammenfassung: | <jats:title>Abstract</jats:title><jats:p>The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse biologic processes in all domains of life. Consequently, there is a great demand for time-resolved data on 2-OG fluctuations that can’t be adequately addressed using established methods like mass spectrometry-based metabolomics analysis. Therefore, we set out to develop a novel intramolecular 2-OG FRET sensor based on the signal transduction protein P<jats:sub>II</jats:sub> from <jats:italic>Synechococcus elongatus</jats:italic> PCC 7942. We created two variants of the sensor, with a dynamic range for 2-OG from 0.1 µM to 0.1 mM or from 10 µM to 10 mM. As proof of concept, we applied the sensors to determine <jats:italic>in situ</jats:italic> glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in <jats:italic>Synechococcus elongatus</jats:italic> PCC 7942 cells and measured 2-OG concentrations in cell extracts from <jats:italic>Escherichia coli in vitro</jats:italic>. Finally, we could show the sensors’ functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.</jats:p> |
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| ISSN: |
2045-2322
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| DOI: | 10.1038/s41598-017-01440-w |